MTC Flashcards
use of cryo-based methods
rapid microscopic analysis
rapid diagnosis
oncologic sx
reasons to perform cryo-based methods
Provide quick gross or microscopic diagnostics to identify an unknown
pathologic process, determine disease extent/margins, detect
metastases, or simply identify a tissue.
* Process tissue to give appropriate and accurate diagnosis and
prognosis, as well as to follow research and particular study protocols.
* Confirm the presence of diseased tissue on permanent sections for
diagnosis.
Reasons not to perform cryo-based methods
The diagnosis of a frozen segment has no immediate ramifications for
decision making.
* Tissue is required for permanent processing (it is unusual or small, or
it requires lengthy research to diagnose).
* Frozen sections are known to exhibit severe artifacts that make
appropriate interpretation difficult.
* Tissue is severely calcified and ossified.
* The possibility of a major infection (e.g. HIV, hepatitis B/C, TB)
arrange
- Tissues are placed on dispensing
slide - Wells are frozen with chuck and
over-chuck - Applying embedding medium
- Tissues are placed in embedding
wells - Block is removed for trimming/
staining - Wells are filled with embedding
media
3 1 4 6 2 5
While awaiting the stain line,
prepared slides should be
immediately immersed in formal
alcohol, 95% alcohol (methanol /
ethanol), or formalin; if this step is
skipped, ___ artifacts will occur
drying
use of IHC
tumor
IHC flowchart
deparaffinization
rehydration
Ag retrieval
blocking
primary antibody
secondary antibody
chromogen application
counterstain
For epitopes which may lose antigenicity with heat, may destroy
epitopes and tissue morphology
enzyme digestion
used method For some drugs such as bleomycin, daunomycin and pepleomycin
oxidizing
used to Minimize contamination of sections
detergent
Background staining can be caused by:
- nonspecific antibody binding - more common in polyclonal antibodies, and
- endogenous peroxidase activity - more problematic in tissues with a high
concentration of hematopoietic components, (e.g. bone marrow) - Nonspecific antibody binding can be reduced by:
> Preincubating the secondary antibody with normal serum from the same species or with
a commercially available universal blocking agent (e.g. goat serum).
targets a single epitope; tends to be more specific
monoclonal antibody
can bind many different epitopes; tends to be more sensitive
polyclonal antibody
function of the secondary antibody
visualize the antigen-antibody reaction
How can nonspecific antibody binding be reduced
preincubating the secondary antibody with normal serum from the same species or with a commercially available universal blocking agent
